Selective restriction fragment amplification:  fingerprinting

ABSTRACT

The invention relates to a process for the controlled amplification of at least one part of a starting DNA containing a plurality of restriction sites for a determined specific restriction endonuclease, and of which at least part of its nucleic acid is unknown. Application of this process to human, animal or plant DNA fingerprinting, to identification of restriction fragment length polymorphisms. Kit for the application of the process.

This application is a divisional of application Ser. No. 08/769,450,filed Dec. 19, 1996, which is a divisional of application Ser. No.08/180,470, filed Jan. 12, 1994, which is a continuation of applicationSer. No. 07/950,011, filed Sep. 24, 1992.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to applications of DNA fingerprinting and the useof DNA markers in a number of different fields including, but notlimited to, plant and animal breeding, variety or cultivaridentification, diagnostic medicine, disease diagnosis in animals andplants, identification or genetically inherited disease in humans,family relationship analysis, forensic analysis, and microbial typing.

More specifically, this invention relates to methods for DNAfingerprinting and for detecting specific DNA markers in genomes rangingfrom microorganisms to higher plants, animals and humans. The inventionalso relates to synthetic DNA molecules and products based thereon whichare used in the methods of the invention in the different fields ofapplication.

2. Description of the Related Art

1. DNA Fingerprinting

DNA fingerprinting or DNA typing, as well as other methods ofgenotyping, profiling and DNA identification analysis, refer to thecharacterization of either similarities or one or more distinctivefeatures in the genetic make up or genome of an individual, a variety orrace, or a species. The general rule is that the closer the geneticrelationship is, the greater the identity or more appropriate thesimilarity of genomes, and consequently distinctive features in thegenome will be rarer. These similar or distinctive features can berevealed by analyzing the DNA of an organism after cleaving the DNA witha restriction endonuclease. Restriction endonucleases are enzymes whichrecognize short nucleotide sequences, usually 4 to 8 bases in length andcleave the two DNA strands, thereby producing fragments of DNA ofdiscrete length. Because of their high degree of sequence specificity,restriction endonuclease will cleave DNA molecules in a very specificfashion. The result is that a reproducible set of DNA fragments will beproduced. DNA fragments can be fractionated according to their length onporous matrices, or gels, yielding typical banding patterns, whichconstitutes a DNA fingerprint of the organism's genetic makeup.

2. DNA Polymorphisms

When the fingerprints of very closely related species, varieties orraces are compared, the DNA fingerprints can be identical or verysimilar. When differences are observed within otherwise identical DNAfingerprints, such differences are referred to as DNA polymorphisms:these are new DNA fragments which appear in a fingerprint. The DNA issaid to be polymorphic at that position and this novel DNA fragment canbe used as a DNA marker. DNA polymorphisms detected in DNA fingerprintsobtained by restriction enzyme cleavage can result from any of thefollowing alterations in the DNA sequence; mutations establishing therestriction endonuclease target site, mutations creating new targetsites, insertions, deletions or inversions between the two restrictionsites.

Such DNA polymorphisms are generally referred to as RFLP, RestrictionFragment Length Polymorphisms. Such mutual changes will behave as bonafide genetic markers when they are inherited in a mendelian fashion.Consequently, DNA polymorphisms can be used as genetic markers in muchthe same way as other genetic markers in parentage analysis, in geneticstudies on the inheritance of traits, or in the identification ofindividuals.

3. DNA Fingerprinting Techniques

For almost all living organisms, except viruses, restriction digests ofthe total genomic DNA of the organisms yield so many bands that it isnot possible to score individual bands. Therefore, all methods for DNAfingerprinting are based on the principle that only a small fraction ofthe DNA fragments are visualized so as to yield a simple banding patternwhich constitutes the DNA fingerprint.

The most widely utilized method involves digesting the DNA of theorganism with restriction endonucleases, fractionating the restrictionfragments by gel electrophoresis, transferring and binding thefractionated DNA fragments onto membranes and hybridizing the membranewith a specific DNA fragment (“probe”). The DNA fragment will formdouble-stranded DNA molecules with the DNA fragment (or fragments) onthe membrane which has (have) complementary nucleotide sequences. Whenthe probe is tagged with a visualizable marker, the DNA fragment towhich the probe is attached can be visualized. This procedure isgenerally referred to as “Southern hybridization”. When differences areobserved in the sizes of the corresponding restriction fragments towhich the probe attaches in closely related genomic DNA molecules, thesedifferences are referred to as DNA polymorphisms, more specificallyrestriction fragment length polymorphisms. The restriction fragmentlength differences correspond to the different allelic forms of thegenetic locus recognized by the DNA probe. Although the Southernhybridization method for DNA fingerprinting has been widely used, themethod is laborious and time consuming.

Furthermore, the method has a low resolution and can thus only be usedto score single loci or a few loci at most in a single reaction.

4. Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) technique is a method forsynthesizing specific DNA fragments in vitro. The method relies on theuse of specific oligonucleotides, which will attach to unique sequenceson a DNA molecule and a thermostable DNA polymerase. Theoligonucleotides are designed in such a way that they can anneal to theopposite strands of the DNA and serve as primers in a DNA synthesisreaction in such a way that each will direct the synthesis of new DNAstrands. Hence, in one round of synthesis a complete copy of the DNAmolecule between the primers will be made, so that the DNA between theprimers is duplicated. Each round of DNA synthesis results in thedoubling of the amount of DNA, hence leading to the amplification of theDNA comprised between the two primers. Consequently, the PCR techniqueallows one to synthesize a precise DNA segment using a small amount of“substrate DNA”.

SUMMARY OF THE INVENTION

In the present invention we have devised a new method to amplify, withthe PCR method, restriction fragments obtained after cleaving the DNA ofan organism with at least one restriction enzyme. In this novelapplication of the PCR method the oligonucleotides used are not directedagainst a known DNA sequence but are designed such that they recognizethe ends of the restriction fragments. To this end it is to modify theends of the restriction fragments by adding oligonucleotide linkers (oradaptors) to the ends. The reason for this is that the ends ofrestriction enzymes have only usually few nucleotides in common, i.e. 2to 8 nucleotides, too short to be used to design primers for PCRamplification.

The invention is based on the use of a novel application of polymerasechain reaction technique (PCR) for amplifying one or more restrictionfragments from complex mixtures of DNA fragments obtained by digestinggenomic DNA molecules with restriction endonucleases. One particularadvantage of the invention is to enable the amplification of DNArestriction fragments in situations where the nucleotide sequence of theends of the restriction fragments are not determined. In such cases theusual sequence specific primers hybridizing to each strand of arestriction fragment to be amplified can not be defined and thereforeone cannot use the methods known in the art for amplification purposes.

The method of the invention can be used for instance in two differentways, leading to two different types of applications:

-   -   (1) Methods for DNA fingerprinting of genomes by randomly        selecting subsets of one or more restriction fragments to be        amplified by the PCR technique. The invention also covers        synthetic oligonucleotides for use in said methods and some        applications of said methods can be forensic typing, microbial        identification, varietal identification, pedigree analysis and        screening of DNA markers linked to genetic traits;    -   (2) Methods for identifying one or more preselected DNA        fragments which can be polymorphic, by PCR amplification. The        Invention also covers specific synthetic oligonucleotides for        use in said methods and some applications of said methods can be        the screening of genetically inherited diseases in humans,        monitoring the inheritance of agronomic traits in plant and        animal breeding and the detection of infections agents in        diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a graphic outline for obtaining tagged restrictionfragments by digesting genomic DNA molecules with a restriction enzymeand subsequent ligation of adaptors.

FIG. 2 depicts the ligation of adaptors to different ends of restrictionfragments: flush ends and staggered ends.

FIG. 3 (SEQ ID NOS:68-72) depicts the PCR amplification of taggedrestriction fragments. The boxed areas depict the adaptors which areligated to the restriction fragment, and the primers which are used inthe PCR amplification. The arrows indicate the direction of DNAsynthesis.

FIG. 4 provides a graphic outline for the PCR amplification of taggedrestriction fragments.

FIG. 5 (SEQ ID NOS:73-78) shows the general design of the selectiveprimers used in the PCR amplification or tagged restriction fragments.The boxes denote the constant sequences at the ends of the restrictionfragments. The selectivity of the primers is illustrated in two exampleswhere there is respectively a perfect match and a total mismatch betweenthe selective base sequence and that of the restriction fragmenttemplate DNA.

FIG. 6 (SEQ ID NOS:79-83) shows the principle of selective PCRamplification using a PCR primer which selects template DNA moleculeshaving a trinucleotide sequence adjacent to the adaptor sequence.

FIG. 7 depicts the selective PCR amplification of tagged restrictionfragments.

FIG. 8 (SEQ ID NOS:84-89) shows the principle of fragment specificamplification using a combination of two PCR primers each comprising 4selective bases. Each primer forms a double-stranded structure in thedifferent strand of the restriction fragment and thereby forms aprimer/template complex from which DNA synthesis can be initiated(represented by the arrows).

FIG. 9 (SEQ ID NO:90) depicts the general sequence elements which arerecognized with the method of selective restriction fragmentamplification (SFRA), including the two nucleotide sequences which arerecognized and the distance separating the two sequences.

FIG. 10 depicts the types of nucleotide sequence variations which aredetected in the method or identifying amplified fragment lengthpolymorphisms.

FIG. 11 shows a 1.0% agarose gel with the analysis of the results of theamplification of Tomato DNA restricted with PstI, using primers ofincreasing selectivity.

FIG. 12 shows a 1.0% agarose gel with the analysis of the results ofspecific amplification of 3 different PstI fragments of Tomato DNA usingfragment specific primers.

FIG. 13 shows a 2.5% polyacrylamide/1% agarose gel with DNA fingerprintsobtained by Selective Restriction Fragment Amplification of two Tomatolines.

FIG. 14 shows part of a 4.5% denaturing polyacrylamide gel with DNAfingerprints of 4 Tomato lines using SRFA with the enzyme combinationPstI/MseI.

FIG. 15 shows part of a 4.5% denaturing polyacrylamide gel with DNAfingerprints of 10 Lactuca lines using SRFA with the enzyme combinationPstI/MseI.

FIG. 16 shows part of a 4.5% denaturing polyacrylamide gel with DNAfingerprints of 2 Corn lines using SRFA with the enzyme combinationsPstI/TaqI and EcoRI/TaqI.

FIG. 17 shows part of a 4.5% denaturing polyacrylamide gel with DNAfingerprints of 26 Xanthomonas campestris strains using SRFA with theenzyme combination ApaI/TaqI.

FIG. 18 shows part of a 4.5% denaturing polyacrylamide gel with DNAfingerprints of different individuals or 4 domestic animals Chicken,Pig, cow and horse using SRFA with the enzyme combination SccI/MseI.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

In the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

-   -   Restriction Endonuclease: a restriction endonuclease or        restriction enzyme is an enzyme that recognizes a specific base        sequence (target site) in a double-stranded DNA molecule, and        will cleave both strands of the DNA molecule at every target        site.    -   Restriction Fragments: the DNA molecules produced by digestion        with a restrictive endonuclease are referred to as restriction        fragments. Any given genome will be digested by a particular        restriction endonuclease into a discrete set of restriction        fragments. The DNA fragments that result from restriction        endonuclease cleavage are separated and detected by gel        electrophoresis.    -   Restriction fragment Length Polymorphism (RFLP): the genomic DNA        of two closely related organisms, for example, will exhibit        differences in their nucleotide sequence composition at many        sites. When these differences occur in the target site for a        restrictive endonuclease, the modified target site will not be        cleaved at that point. Likewise, a nucleotide sequence variation        may introduce a novel target site where none exists in the other        organism, causing the DNA to be cut by the restrictive enzyme at        that point. Alternatively, insertions or deletions of        nucleotides occurring in one organism between two target sites        for a restriction endonuclease will modify the distance between        those target sites. Because of this, digestion of the two        organism's DNA will produce restriction fragments having        different lengths. A polymorphism in the length of restriction        fragments produced by digestion of the DNA of the two organisms        will result.    -   Gel Electrophoresis: To detect restriction fragments, an        analytical method for fractionating double-stranded DNA        molecules on the basis of size is required. The most commonly        used technique for achieving such fractionation is gel        electrophoresis. The rate at which DNA fragments move in such        gels depends on their size; thus, the distances traveled        decrease as the fragment lengths increase. The DNA fragments        fractionated by gel electrophoresis can be visualized directly        by a staining procedure if the number of fragments included in        the pattern is small.    -   Synthetic oligonucleotides: the single-stranded DNA molecules        having preferably from almost 10 to almost 50 bases, which can        be synthesized chemically are referred to as synthetic        oligonucleotides. In general, these synthetic DNA molecules are        designed to have a unique nucleotide sequence, although it is        possible to synthesize families of molecules having related        sequences and which have different nucleotide compositions at        specific positions within the nucleotide sequence. The term        synthetic oligonucleotides will be used to refer to DNA        molecules having a unique nucleotide sequence. The term mixed        synthetic oligonucleotides will be used to refer to families of        related synthetic oligonucleotides.    -   Ligation: the enzymatic reaction catalyzed by the enzyme ligase        in which two double-stranded DNA molecules are covalently joined        together is referred to as ligation. In general, both DNA        strands are covalently joined together, but it is also possible        to prevent the ligation of one of the two strands, through        chemical or enzymatic modification of one of the ends. In that        case the covalent joining will occur in only one of the two DNA        strands.    -   Adaptors: short double-stranded DNA molecules, with a limited        number of base pairs, e.g. 10 to 30 base pairs long, which are        designed in such a way that they can be ligated to the ends of        restriction fragments. Adaptors are composed of two synthetic        oligonucleotides which have nucleotide sequences which are in        part complementary to each other. When mixing the two synthetic        oligonucleotides, they will form a double stranded structure in        solution under appropriate conditions, one of the ends of the        adaptor molecule is designed so that it can be ligated to the        end of a restriction fragment, the other end is designed so that        it cannot be ligated.    -   Polymerase Chain Reaction (PCR): the enzymatic reaction in which        DNA fragments are synthesized from a substrate DNA in vitro is        referred to as PCR. The reaction involves the use of two        synthetic oligonucleotides, which are complementary to        nucleotide sequences in DNA molecules which are separated by a        short distance of a few hundred to a few thousand base pairs,        and the use of a thermostable DNA polymerase. The chain reaction        consists for example of a series of 10 to 30 cycles. In each        cycle the substrate DNA is first denaturated at high        temperature. After cooling down the synthetic oligonucleotides        which are present in vast excess will form double-stranded        structures with the substrate DNA molecules in solution at        specific sites on the substrate DNA molecule that have        complementary nucleotide sequences. The        oligonucleotide-substrate DNA complexes will then serve as        initiation sites for the DNA synthesis reaction catalyzed by the        DNA polymerase, resulting in the synthesis of a new DNA strand        complementary to the substrate DNA strand.    -   DNA amplification: the term DNA amplification will be used to        denote the synthesis of double-stranded DNA molecules in vitro        using Polymerase Chain Reaction (PCR). The products of the PCR        reaction will be referred to as amplified DNA fragments.    -   Primers: in general, the term primer refers to a DNA strand        which can prime the synthesis of DNA. DNA polymerase cannot        synthesize DNA de novo without primers. DNA polymerase can only        extend an existing DNA strand in a reaction in which the        complementary strand is used as a template to direct the order        of nucleotides to be assembled. We will refer to the synthetic        oligonucleotide molecules which are used in the PCR reaction as        primers.    -   Southern Hybridization Procedure: the purpose of the Southern        hybridization procedure, also referred to as Southern blotting,        is to transfer physically DNA fractionated by agarose gel        electrophoresis onto a support such as nylon membrane or        nitrocellulose filter paper while retaining the relative        positions of DNA fragments resulting from the fractionation        procedure. The methodology used to accomplish the transfer from        agarose gel to the support is to draw the DNA from the gel into        the support by capillary action.    -   Nucleic Acid Hybridization: Nucleic acid hybridization is used        to detect related DNA sequences by hybridization of        single-stranded DNA on supports such as nylon membrane or        nitrocellulose filter papers. Nucleic, acid molecules that have        complementary base sequences will reform the double-stranded        structure if mixed in solution under the proper conditions. The        double-stranded structure will be formed between two        complementary single-stranded nucleic acids even if one is        immobilized on a support. In the Southern hybridization        procedure, the latter situation occurs.    -   Hybridization Probe: to detect a particular DNA sequence in the        Southern hybridization procedure, a labelled DNA molecule or        hybridization probe is reacted to the fractionated DNA bound to        a support such as nylon membrane or nitrocellulose filter paper.        The areas on the filter that carry DNA sequences complementary        to the labelled DNA probe become labelled themselves as a        consequence of the reannealing reaction. The areas of the filter        that exhibit such labelling can then be detected according to        the type of label used. The hybridization probe is generally        produced by molecular cloning of a specific DNA sequence form        the maize genome.

This invention relates more particularly to a process and means whichenable the polymerase chain reaction (PCR) be applicable to thedetection of restriction fragment polymorphisms (RFPs) including lengthpolymorphisms. This invention comprises methods for detecting RFPs,synthetic oligonucleotides for use in the methods of the invention, kitscomprising means for detecting RFP's, and applications of the methodsand procedures of the invention for plant and animal breeding,diagnostics of genetically inherited diseases, identification oforganisms, and forensic typing, etc. . . .

Specifically, this invention provides means for the identification ofeither individual genomic restriction fragments or of sets of genomicrestriction fragments from any organism, microorganism, plant, animal orhuman, which are either individually genetically linked to one or moreparticular traits or that collectively provide a fingerprint of thegenuine that can be used to identify an organism, a variety or anindividual.

The general method of the invention for production and foridentification of restriction fragments involves the use of restrictionendonucleases, ligation of synthetic olignucleotides to the restrictionfragments, and PCR amplification of restriction fragments. Restrictionendonucleases cleave genomic DNA molecules at specific sites, targetsites, thereby generating restriction fragments.

PCR amplification of restriction fragments no matter whether one knowsthe nucleotidic sequence of the ends of the restriction fragments ornot, can be achieved according to the invention, by first ligatingsynthetic oligonucleotides (adaptors) to the ends of restrictionfragments, thus providing each restriction fragment with two common tagswhich will serve as a anchor base for the primers used in PCRamplification.

Typically, restriction enzymes produce either flush ends, in which theterminal nucleotides of both strands are base paired or staggered endsin which one of the two strands protrudes to give a short single strandextension (FIG. 2). In the case of restriction fragments with flushends, adaptors are used with one flush end. In the case of restrictionfragments with staggered ends adaptors are used which have a singlestranded extension which is complementary to the single strandedextension of the restriction fragment. Consequently, for each type ofrestriction fragment specific adaptors are used, which differ in one ofthe ends so as to allow the adaptor to be ligated to the restrictionfragment. Typically, the adaptors used are composed of two syntheticoligonucleotides which are in part complementary to each other, andwhich are usually approximately 10 to 30 nucleotides long, preferably 12to 22 nucleotides long and which form double-stranded structures whenmixed together In solution. Using the enzyme ligase the adaptors areligated to the mixture of restriction fragments. When using a largemolar excess of adaptors over restriction fragments one ensures that allrestriction fragments will end up carrying adaptors at both ends.Restriction fragments prepared with this method will be referred to astagged restriction fragments and the method will be further referred toas restriction fragment tagging.

The adaptors can now serve as templates for the primers having thecharacteristics here above defined used in the subsequent PCRamplification reaction. In a preferred embodiment of the invention, therestriction fragment carries the same adaptor at both of its ends and asingle PCR primer can be used to amplify the restriction fragment asillustrated in FIG. 3. Since in such a case all restriction fragmentsare tagged in the same way, it is obvious that PCR amplification of amixture of tagged restriction fragments will amplify all restrictionfragments in a synchronous fashion. In another embodiment using twodifferent restriction enzymes to cleave the DNA, two different adaptorsare ligated to the ends of the restriction fragments. In this case twodifferent PCR primers can be used to amplify such restriction fragments.In another preferred embodiment using two different restriction enzymesthe adaptor for one of the enzyme ends is biotinylated. This allows oneto select out of the complex mixture of restriction fragments thoserestriction fragments carry at least one end for this restrictionenzyme, using usual methods for isolating biotinylated molecules. Thisstep reduces the complexity of the starting mixture of restrictionfragments and constitutes an enrichment step prior to the PCRamplification, thereby reducing in certain instances the background. Thesimultaneous amplification of several different fragments is oftenreferred to as multiplex PCR. The principle of multiplex restrictionfragment amplification is illustrated in FIG. 4

The present invention is further based on the definition of specificallydesigned primers and specific methods to direct the PCR amplificationreaction in such a way that a controlled amplification is possible andin a particular embodiment of the invention, in such a way that only asmall subset of tagged restrictive fragments is amplified.

In general, restriction endonuclease digests of genomic DNA, and inparticular of animal, plant or human genomic DNA, yields very largenumbers of restriction fragments. The number of restriction fragmentsdepends upon the size of the genome and of the frequency of occurrenceof the target site of the restriction endonuclease in the genome, whichin turn is primarily determined by the number of nucleotides in thetarget site. The number of nucleotides in the target sites of commonlyused restriction endonucleases ranges from 4 to 8. The genome sizes oforganisms vary widely from a few million base pairs in the case ofmicroorganisms to several billion base pairs for animals and plants.Hence, the number of restriction fragments obtained after cleavinggenomic DNA molecules with a restriction enzyme can vary from a fewhundred to several million. Generally, the number of restrictionfragments is so large that it is not possible to identify individualrestriction fragments in genomic DNA digests fractionated by gelelectrophoresis. Such digests usually produce a smear of bands.

PCR amplification of tagged restriction fragments should thus alsoproduce a smear of bands since all fragments should coamplifysynchronously in the PCR reaction. In a preferred embodiment of theinvention applicable to genomic DNAs of large sizes, we have used ageneral principle to limit the number of restriction fragments which areto be amplified. This is done by preselecting a subset of taggedrestriction fragments so that only a relatively small number of taggedrestriction fragments will be amplified during the PCR amplificationreaction.

The selective principle defined in this embodiment of the inventionresides in the design of the oligonucleotides which are used as primersfor the PCR amplification, as is illustrated in FIG. 5.

Tagged restriction fragments have the following general structure: avariable DNA sequence (corresponding to the restriction fragment beforetagging), flanked on both aides by a constant DNA sequence. The invertedDNA sequence (constant DNA sequence) is composed of part of the targetsequence of the restriction endonuclease and of the sequence of theadaptor attached to both ends of the restriction fragment. The variablesequences of the restriction fragments comprised between the ConstantDNA sequences are usually unknown, and will thus have a random sequencecomposition. Consequently, the nucleotide sequences flanking theconstant DNA sequence will be totally random in a large mixture ofrestriction fragments.

The present invention therefore also provides specific PCR primers whichcomprise a constant nucleotide sequence part and in the embodiment ofthe invention relying to the amplification of a restricted subset of therestriction fragments obtained, a variable sequence part. In theconstant sequence part the nucleotide sequence is designed so that theprimer will perfectly base pair with the constant DNA sequence of one ofthe DNA strands at the end of the restriction fragment. The variablesequence part comprises a randomly chosen nucleotide sequence rangingfrom 1 to 10 bases chosen.

The expression “variable sequence” more exactly designates a sequenceconsisting of selected nucleotides forming a sequence which will thenremain constant for the purpose of amplifying a subset of restrictionfragments. In a particular embodiment of the invention, severalsequences of selected bases can be used, in order to define several,distinguished primers. In such a case, primers can have the sameconstant sequence and variable sequences made of selected bases whichare different among the primers thus formed.

It is the addition of these variable (selected) sequences to the '3 endof the primers which will direct the preselection of tagged restrictionfragments which will be amplified in the PCR step; when the PCR reactionis performed under appropriate conditions the primers will only initiateDNA synthesis on those tagged restriction fragments in which thevariable DNA sequence can perfectly base pair with the template strandof the tagged restriction fragment, as illustrated in FIG. 5.

The selection is determined by the number of nucleotides residing in thevariable sequence part of the primer: the selectively of the primersincreases with the number of nucleotides in the variable (selected)sequence part. We will also use the term selective bases to denote thenucleotides in the variable sequence part thus showing that theselection of these bases renders the primer selective. It must berealized that a tagged restriction fragment will only be amplified whenthe selective bases of the primers used recognize both complementarysequences at the ends of the fragment. When the primer matches with onlyone end, the amplification will be linear rather than exponential, andthe product will remain undetected.

It is possible to estimate beforehand the degree of selectivity obtainedwith variable sequences with different numbers of selective bases, usingthe general formula 4^(2n), where n equals the number of selectivebases; using 1 selective base, 1 out of 16 tagged fragments will beamplified, using 2 selective bases, 1 out of 256, using 3 selectivebases, 1 out of 4.096, using 4 selective bases, out of 65.536, and soon, will be amplified. One preferred embodiment of the present inventionthus; allows one to selectively amplify a random subset of taggedrestriction fragments from any genomic DNA digest regardless of thenumber or fragments produced by the restriction enzyme used.

In a preferred embodiment, the number of selective nucleotides is chosenso that the number of restriction fragments which will be amplified islimited to 5 to 200. Although this number can be calculated by dividingthe number of fragments by 4^(2n), a precise prediction is not possiblebecause not all restriction fragments can be amplified with equalefficiency. Hence, in practice, one finds less fragments of theamplification than theoretically expected. It should also be pointed outthat mixtures of two (or more) primers can be used. This will allow theamplification of the fragments recognized by each primer and inaddition, the fragments recognized by the two primers. Finally, itshould be pointed out that the selection based on the base pairingbetween the selective nucleotides of the primer and the complementarytemplate is strongly influenced by the temperature chosen for theannealing step in the PCR reaction when this temperature is below or tooclose to the melting temperature of the primer/template complex, primerswill anneal the imperfectly matching template sequences allowing amismatch to occur in the complex. This should be avoided because itwill, lead to the amplification of many more fragments than predicted,producing more variable results.

The PCR products obtained in accordance with the invention can beidentified using standard fractionation methods or separating DNAmolecules according to size followed by staining of the DNA moleculeswith appropriate agents. Alternatively, the primers used for the PCRamplification can be tagged with a suitable radio-active labelled orfluorescent chromophore thus allowing the identification of the reactionproducts after size fractionation. In a preferred embodiment of theinvention the PCR products are fractionated by gel electrophoresis usingstandard gel matrices such as, but not limited to, agarose,polyacrylamide or mixed agarose/polyacrylamide. The PCR productsobtained according to the invention will be denoted further by the termAmplified Restriction Fragments (ARF).

The means and method of the present invention can be used to generatesets of ARF from restriction digest of any complex genome. The inventionpermits the number of restriction fragments obtained to be tuned inaccordance with the resolution of the gel fractionation system used toseparate the ARFs. In one particular embodiment the selective primersare designed to produce 5 to 10 ARFs which are then separated by agarosegel electrophoresis. Another particular embodiment involves the use ofselective primers which are designed to produce 20 to 50 ARFs which arethen separated on a high resolution gel electrophoresis system such as,but not limited to, polyacrylamide gels or mixed polyacrylamide-agarosegels.

In one preferred embodiment the restriction enzyme or enzymes are chosento yield restriction fragments in the size range of 20 to 1000 basepairs, because as is generally known for PCR amplification, thisfragment size range is amplified most effectively. Although muchfragments can be fractionated on various standard gel matrices, bestresults are obtained by fractionation on denaturating polyacrylamide gelsystems as are currently used for DNA sequencing.

In accordance with the invention, different sets of ARFS are obtainedwith each different selective primer in the PCR amplification reaction.The patterns of ARFs identified after separation constitute unique andperfectly reproducible fingerprints of the genomic DNA. Suchfingerprints can have several applications such as, but not limited to,forensic typing, the diagnostic identification of organisms, and theidentification of species, races, varieties or individuals. The level ofidentification will be determined by the degree of similarity (thedegree of variability) exhibited by different members of a specificgroup. The viability or similarity is determined by the degree ofvariation in the nucleotide composition of the related genomes. Theunderlying principle of the invention is that in each AmplifiedRestriction fragment two nucleotide sequences are detected which areseparated from each other by a given distance, as is illustrated in FIG.9. Each of the two nucleotide sequences is composed of two parts: (a)the target site for the restriction endonuclease and (b) the nucleotidesequence adjacent to the target site which is included in the selectiveprimer. In related organisms, species, varieties, races or individualsthese sequence elements and their relative distances will be conservedto a greater or lesser degree. Hence, the fingerprints constitute abasis for determining the degree of sequence relationships betweengenomes. On the other hand, differences in the ARF patterns can be usedto distinguish genomes from each other. The particular advantages of thepresent invention over other methods for fingerprinting genomes is thehigh resolution that can be obtained with the method: several tens oreven hundreds of ARFs can be compared simultaneously.

Another particular application of the present invention involves thescreening and identification of restriction fragment polymorphisms(RFP). Changes in the nucleotide composition of genomic DNA often resultin polymorphisms of restriction fragments: insertions or deletionsaffect the size of the restriction fragments containing them (FIG. 10),nucleotide changes can result in the elimination of restrictionendonuclease target sites or the creation of new restrictionendonuclease target sites. The most commonly used techniques foridentifying such changes are Southern blotting experiments using clonedDNA probes, a technique usually referred to as restriction fragmentlength polymorphism (RFLP) detection. This technique involves theextensive screening of randomly cloned DNA fragments in Southernblotting experiment: for associated RFLPs among different genomes. Inaccordance with the method of the present invention, RFPs can beidentified directly by comparing the ARFs obtained from differentgenomes. In principle, the method of the present invention is moresensitive for detecting RFPs because not only differences in the targetsites of the restriction endonuclease are detected, but also differencesin the adjacent nucleotide sequences comprised in the selective PCRprimers. Consequently, the method of the present invention constitutes afar superior method for detecting RFPs.

RFLPs are now currently used for several applications including forensictyping, monitoring of genetically inherited diseases in humans andmonitoring the inheritance of agronomic traits in plant and animalbreeding. The underlying principle is that certain DNA polymorphismswhich are closely linked with specific genetic traits can be used tomonitor the presence or absence of specific genetic traits.

According to the method of the present invention, the analysis of ARFpatterns can be used to define the genetic linkage of polymorphic ARFswith specific genetic traits. Such polymorphic ARFs will be furtherreferred to as Amplified Fragment Length Polyrmorphisms (AFLPs) todistinguish them from RFLP type DNA polymorphisms detected in Southernblotting experiments using cloned DNA probes.

One particular application of the present invention involves thedetection of AFLPs linked to specific genetic traits. The applicationinvolves the analysis of ARF patterns obtained with different selectiveprimers in restriction digests of genomic DNA of closely relatedindividuals exhibiting differences in the specific genetic trait and theuse of analysis techniques that can find correlations between theinheritance of one or more AFLPs and the phenotype exhibited by thespecific genetic traits.

A second preferred embodiment of the present invention involves the useof the method of the invention to identify one or more specificrestriction fragments. One specific restriction fragment can beamplified from a complex mixture or tagged restriction fragments byfirst determining the nucleotide sequence of the first 8-12 bases ateach end of the restriction fragment. Based on these sequences one candesign two primers with each 5 to 10 selective nucleotides exhibiting asequence complementary to that of the sequence flanking the restrictionsite of the complementary strand or the restriction fragment. Using suchsets of primers one can obtain, after PCR amplification, a singleamplified fragment. The restriction fragment used in this method can beeither a cloned restriction fragment or an amplified restrictionfragment. Since not many restriction fragments cannot be amplified veryefficiently, the preferred method of the invention for identifyingpolymorphic DNA markers involves first amplifying randomly chosen set offragments and identifying AFLPs which yield strong bands after PCRamplification. These AFLPs can be characterized by sequencing to developrestriction fragment specific primers. Typically, the AFLPs will beisolated by cutting out the corresponding DNA band from the gel, anddetermining the nucleotide sequences at both ends to establish thesequence of the first 5 to 10 nucleotides adjacent to the restrictionendonuclease target sites. Once these nucleotide sequences are known,restriction fragment specific primers can be designed which will onlyamplify a single restriction fragment from a genomic DNA digest. In thisparticular embodiment of the invention, one set of two differentselective primers can be used for detecting a specific restrictionfragment. In each of the two selective primers of one set the selectivebases are chosen such that they are complementary to the nucleotidesequence adjacent to the restriction endonuclease target site, as isillustrated in FIG. 8. The number of selective bases to be included ineach primer depends upon the complexity of the restriction endonucleasefragment mixture.

The PCR technique has developed tremendously over the past few years andis rapidly becoming one of the most widely used diagnostic method inhuman health care. Its application includes amongst others detection ofinfectious diseases and detection of genetically inherited diseases.Each diagnostic test is based on the use of two specific syntheticoligonucleotides which are used as primers in the PCR reaction to obtainone or more DNA fragments of specific lengths. In disease detection thetest will detect the presence of as little as one DNA molecule persample, giving the characteristic DNA fragments. In the case ofgenetically inherited diseases the primers are designed such that theirproducts can discriminate between normal, and disease alleles. Thedistinction either relies on sequence differences in the DNA segment inthe genome which is complementary to the primer or, on distancedifferences between the two primers.

Because the primers exhibit an extremely high degree of specificity, itis possible to monitor different diseases simultaneously, a method oftenreferred to as multiplex PCR. The multiplex PCR method, however, suffersfrom the limitation that generally only few, 5 to 8, different traitscan be monitored simultaneously. The scientific basis for thislimitation is that the optimal conditions for PCR amplification(annealing temperature, Mg+ concentration, primer concentration) varyconsiderably depending on the pair of primers used. In multiplex PCRcompromise conditions have to be established under which all primerpairs yield detectable products. In addition, superimposed upon thisphenomenon there is the phenomenon of strong differences in theefficiency of amplification of different fragments. Consequently, oneoften has encountered the problem that products of certain primer pairsare not detectable in multiplex PCR reactions.

The methods of the present invention in essence overcomes theselimitations of multiplex PCR, because all the primers used in thepresent invention have a substantial part of their nucleotide sequencein common. Furthermore, by selecting AFLPs, we select DNA markers thatare amplified with equal efficiency. Hence, the optima of the PCRamplification conditions for the different selective primers exhibitmuch less variation than is observed with commonly used sequencespecific primers. In essence, ideal compromise between the number ofbases in the synthetic oligonucleotide which are necessary to obtain therequired specificity of detecting a single DNA fragment or a given sizein a complex genome, which is calculated above, and the length andcomposition of the oligonucleotide which is optimal for efficient PCRamplification. The method of the invention thus provides a far superiormethod for multiplex PCR.

The present invention provides a general method for isolating DNAmarkers from any genome and for using such DNA markers in all possibleapplications of DNA fingerprinting.

The following examples and figures provide an illustration of theinvention which is nevertheless not limited to these examples.

EXAMPLES Example 1 Selective Restriction Fragment Amplification ofTomato DNA Using PstI A) Isolation and Modification of the DNA

Total Tomato DNA (Lycopersicon esculentum c.v. Moneymaker) was isolatedfrom young leaves as described by Bernatzski and Tanksley (Theor. Appl.Genet. 72, 314-321). The typical yield was 50-100 μg DNA per gram offresh leaf material. The DNA was restricted with PstI (Pharmacia) anddouble-stranded (ds) PstI-adapters were ligated to the restrictionfragments following the procedure described below. These adapters hadthe following structure (SEQ ID NOS:1-2):

5- CTCCTAGACTGCGTACATGCA -3 3- CATCTGACGCATGT -5The 3′TGCA-overhang in these adapters anneals to the staggered endscreated by PstI. The PstI recognition sequence CTGCAG is not restoredupon ligation of this adapter, because the 5′ C-residue is replaced byA. The ligation reaction was designed in such a way that the end resultis almost exclusively DNA fragment-to-adapter molecules. This wasachieved by: 1. using non-phosphorylated adapters, which excludesadapter-to-adapter ligation, 2. Performing the ligation and restrictionreduction at the same time. The latter procedure results in restrictionof any fragment-to-fragment ligation product, thereby eliminating theseproducts almost completely. Adapter-to-fragment ligation products cannotbe restricted by the restriction enzyme, because the PstI recognitionsequence is not restored in the products. The reaction conditions usedfor the adapter ligation were:

2 μq Tomato DNA

0.2 μg adaptors

20 units PstI

1 unit T4 DNA-ligase

10 μM Tris.HAc pH 7.5, 10 mM MgAc, 50 mM KAc,

2 mM dithiotreitol, 0.5 mM ATP

The ligation reaction was performed in a reaction volume of 20 μl for 3hours at 37° C. After the adapter ligation, non-ligated adapters wereremoved by selective precipitation. For this purpose the reactionmixture was increased to 100 μl and NH4Ac was added to a finalconcentration of 2.5 M. 100 μl ethanol of −20° C. was added and themixture was incubated for minutes at room temperature. The DNA wascollected by centrifugation for 10 minutes at 14000 rpm in a cooledeppendorf centrifuge at 4° C. The DNA pellet was washed once with 0.5 mlor 70% ethanol at room temperature, and dissolved in 40 μl of T0.1E (10mM Tris.HCl pH 8.0, 0.1 mM EDTA). The DNA was stored at −20° C. Theselective precipitation procedure described here removes the non-ligatedadapters efficiently from the reaction mixture, but small DNA-fragments(≦200 bp) are also lost.

B) The Amplification Reaction

The DNA prepared above was used as template for amplification of thePstI-fragments. The reaction mixture for the PCR contained:

1 ng template DNA

150 ng primer

1 unit Taq DNA polymerase (Perkin Elmer)

200 μM of all 4 dNTP's

10 mM Tris.HCl pH 8.5, 1.5 mM MgCl₂, 50 mM KCl

H20 to a total volume of 50 μl

The reaction mixture was covered with 20 μl of light mineral oil toprevent evaporation during the amplification reaction. The PCR wasperformed on a Perkin Elmer DNA Thermal Cycler using the following cycleprofile: 1 minute at 94° C., 1 minute at 60° C., a temperature increasefrom 60° C. to 72° C. at a rate of 1° C./5 seconds, and 2′4 minute at72° C. A total of 33 cycles were performed. After the reaction 20 μlchloroform was added, and 10 μl of loading dye, in this case 50% sucrosewith 0.1% w/v of the dye Orange G (Merck). This was then mixed well withthe reaction mixture and briefly centrifuged to separate the organicfase (mineral oil and chloroform) from the reaction mixture supplementedwith the loading dye. 20 μl of this reaction mixture was analysed on a1.0% agarose gel.C) Amplification of Tomato DNA with Primers of Increasing Selectivity

Tomato DNA restricted with PstI and tagged with the PstI-adapter wasamplified using the conditions specified above. Four different primerswere selected with the sequences (SEQ ID NOS:3-6):

1. 5-CTCGTAGACTGCGTACA-3 2. 5-GACTGCGTACAtgcagA-3 3.5-GACTGCGTACAtgcagAC-3 4. 5-GACTGCGTACAtgcagACC-3Primer 1 is part of the top strand of the adapter used to modify theDNA, and therefore should amplify all PstI-fragments. Primer 2 containspart of the adapter sequence, the PstI-recognition sequence (lower caseletters) and one selective nucleotide (bold) and should amplifytheoretically about 1/16 part of all PstI-fragments. Primers 3 and 4 aresimilar to primer 2, but contain 2 and 3 selective nucleotidesrespectively, and therefore are expected to amplify about 1/256 and1/4096 of the PstI-fragments. Part of the reaction mixtures wereanalysed on a 1.0% agarose gel, which is shown in FIG. 11. Lanes 1 and 6of this figure contain DNA markers, of which the sizes are indicated atthe left. Lanes 2, 3, 4 and 5 contain the PCR's obtained with primers 1,2, 3 and 4 respectively. The results indicate that only in case of theprimer with 3 selective nucleotides, the number of amplified fragmentswas such that a clear band pattern was obtained. The other 3 primersgave band patterns, which could not be resolved on agarose gels, becauseto many PCR products were generated. Within these many PCR productsalways some fragments predominate, and are seen as bands on a backgroundsmear of the other PCR products. Probably these stronger products arepresent in higher copy numbers on the Tomato genome, or amplify moreefficient than the other products. It was anticipated that primers with3 selective nucleotides had to be used to generate a clear band patternon aqarose gels, because of the total number of PstI-fragments of Tomatogenomic DNA (20.000 to 100.000).

D) Analysis of Amplified Fragments on Southern Blots

The amplified fragments were tested on Southern blots to verify thatthese fragments corresponded to bona fide restriction fragments of thesame size. For this purpose four individual fragments obtained withprimer 4, were cut out of the agarose gel. The DNA was purified fromthese gel slices by means of absorption to glass beads (Gene Clean,manufacturer Bio 101), and part of the purified DNA was reamplified toobtain about 1 μg of each of the four DNA fragments. The reamplificationreactions were subsequently electrophoresed on a 1.0% preparativeagarose gel, and the desired DNA fragments were purified. 200 ng of eachfragment was labeled with (α-32P)dATP using a random hexamer labellingkit according to procedures advised by the manufacturer (BoehrinqerMannheim). Total Tomato DNA was restricted with PstI, andelectrophoresed on a 1.0% agarose gel. Four clearly separated lanes eachcontaining about 3 μg of restricted DNA were used. Next, the agarose gelwas blotted to a Genescreen+hybridisation membrane as indicated by themanufacturer (New England Nuclear). After blotting the gel was cut infour slices, each containing one lane of the Tomato DNA restricted withPstI. These four slices were each hybridised to one of the four DNAprobes following the procedure described by Klein-Lankhorst et al.(Theor. Apll. Genet. 81, 661-667). The hybridised blots wereautoradiographed for 40 hours using Kodak XAR5 films. The resultsobtained showed that all genomic DNA fragments recognised by the fourDNA probes, had the same length as these probes. This demonstrated thatthe amplified fragments, used as probes, originated from the fragmentsdetected on the blots.

E) Selective Amplification of a Single Restriction Fragment

Three sets of primers were designed for 3 corresponding randomPstI-fragments from Tomato genomic DNA, of which the sequence next tothe PstI-recognition sequence was known. Sets of primers with 5selective nucleotides were made as shown below.

Primer set 1: Sequence 1; (SEQ ID NO: 7)5-ctgcagCAGTACCACC-----CCGGCACCTGctgcag-3 5-TGCGTAACATtgcagCAGTA-33-TGGACgacgtACATGCGT-5 Primer 1.1 (SEQ ID NO: 8) Primer 1.2 (SEQ ID NO:9) Primer set 2; Sequence 2 (SEQ ID NO: 10):5-ctgcagCCGAATCTCT-----AGTGAGTTAGctgcag-3 5-TGCGTACAtgcagCCGAA-33-CAATCgacgtACATGCGT-5 Primer 2.1 (SEQ ID NO: 11) Primer 2.2 (SEQ ID NO:12) Primer set 3: Sequence 1 (SEQ ID NO: 13):5-ctgcagAATACCAAGA-----GCACCACAGctgcag-3 5-TCCCTACAtgcagTTATG-33-GTGTCgacgtACATGCGT-5 Primer 3.1 (SEQ ID NO: 14) Primer 3.2 (SEQ ID NO:15)Tomato DNA was digested with PstI and adapters were ligated to the endsof the restriction fragments as described above. This DNA was used astemplate in PCR's with Primer sets 1 or 2 or 3, using the conditions asdescribed in one of the previous sections. The reaction products of eachPCR were analysed on a 1.0% agarose gel. This gel is shown in FIG. 12.FIG. 12 shows 13 lanes, of which lanes 1, 2, 12 and 13 are DNA markers.The sizes in kilobases of these markers are indicated at both sides ofthe gel. Lanes 3, 6 and 9 show plasmid DNA with each of the threePstI-fragments restricted with PstI, which yields the vector fragment,pUC18 (Yanisch-Perron et al., Gene 33, 103-119), and the insertedPstI-fragment. Lanes 4 and 5 show amplification with primer set 1 of 5fg of the corresponding plasmid DNA and 1 ng of total genomic DNArespectively. Lanes 7 and 8 show amplification with primer set 2 ofplasmid DNA and total genomic DNA, and lanes 10 and 11 showamplification with primer set 3. These results demonstrate that it ispossible to amplify a single PstI-fragments out of a mixture of at least20.000 fragments using the selective restriction fragment amplificationtechnique with primers having 5 selective nucleotides.

F) Identification of DNA Polymers Using SRFA

In the previous sections it was clearly demonstrated that with theselective restriction fragment amplification technique it is possible toamplify restriction fragments, either at random, or specific fragments,when sequence information is available. Hence, it should be possible tosearch for restriction site polymorphisms between two individuals of thesame species. This is described below for two Tomato lines, which arevery related but differ in the presence of the root knot nematoderesistance gene, Mi, in one or the lines. This Mi-gene originates fromLycopersicon peruvianum, a species distantly related to the edibleTomato L. esculentum. It has been introduced into the L. esculentum lineby crossing, and subsequent back crossing 12 times to the L. esculentumparent, and selecting the offspring for presence of the Mi-gene.Therefore, the two Tomato lines differ only in a small portion of theirgenetic material, i.e. the Mi-gene and surrounding region. The Mi-regionwas calculated to constitute <1% of the genome of this line, usingclassical genetic methods.

DNA was isolated from the two Tomato lines (line 83M-71392,Mi-sensitive, and line 83M-71398, Mi-resistant, obtained from De RuiterSeeds, Bleiswijk, The Netherlands) and subsequently restricted with PstIand provided with adapters as described above. A large number ofamplification reactions were performed using primers, which differed intheir extension of selective nucleotides. Three selective nucleotideswere used, and apart form single primers also combinations of twodifferent primers were used. The reactions were analysed on mixedpolyacrylamide/agarose gels: 2.5% polyacrylamide and 1.0% agarose wasused, with a ratio acrylamide to bisacrylamide of 20:1. Gels were run ona Protean II gel unit (Biorad), using spacers of 1.5 mm. A total of 16different primers was used giving 16 reactions with a single primer, and120 reactions with all possible combinations of two primers. A typicalexample of a gel with six of these combinations is shown in FIG. 13.Lanes 1 and 14 of this gel contain DNA markers, of which the sizes inkilobases are indicated at the right side of the gel. Lanes 2 and 3, 4and 5, 6 and 7 etc contain amplifications with a specific primer orprimer pair of the two Tomato lines. The screening for restriction sitepolymorphisms yielded a number a fragments, three of which were veryprominent and which are depicted in lanes 9, 11 and 12 of FIG. 13(indicated by a small circle). The polymorphic bands in lanes 9 and 11were expected to be the same, because the same primer was present inboth reactions (the difference is the presence of a second primer inlane 11). The two polymorphic fragments of lanes 11 and 12 were cut outof the gel, the gel slices were crushed by forcing them through a 18gauge needle and the DNA was eluted from the gel slices by elutionthrough diffusion in 200 μl of 100 mM Tris.HCl pH 8.0, 10 mM EDTA. 2 μlwas used for reamplification of these fragments as described above. 200ng of each fragment was made blunt end using T4 DNA polymerase andsubsequently ligated to 100 ng of plasmid vector pUC18 (Yanisch-Perronet al., Gene 33, 103-119) restricted with SmaI. The ligation mixture wastransformed to E. coli and for each fragment one recombinant E. coliclone was selected for sequence analysis. All these manipulations wereperformed using standard procedures as described by Sambrook, Fritschand Maniatis in: Molecular Cloning, A Laboratory Manual (Cold SpringHarbor Laboratory Press, New York).

Two sets of primers with 6 selective nucleotides were synthesised basedon the sequences of the two fragments as described above. We were ableto amplify each fragment specifically using these primer sets. Fragmentswere only amplified from the Tomato line, from which they originated.Hence, these primer sets exhibited the same polymorphism, initiallyfound with the primers with 3 selective nucleotides used to find thispolymorphism.

Example 2 Selective Restriction Fragment Amplification of Tomato DNAwith Two Restriction Enzymes

In example 1 the principle of selective restriction fragmentamplification (SRDA) is exemplified using Tomato DNA and the restrictionenzyme PstI. In this example SRFA using two different restrictionenzymes, PstI and MseI, will be illustrated.

Isolation and Modification of the DNA

Total Tomato DNA was isolated from young leaves as described inexample 1. Two pairs of so called isogenic lines were used as source ofthe DNA, name Gem^(R) and Gem^(S), and CGR26 and GCR151 respectively(These lines are described in the following references: Denby andWilliams, (1962), Can. J. Plant Sci. 42, 601-685, Smith and Ritchie,(1983), Plant Mol. Biol. Rep. 1, 41-45). The two individuals of eachpair of isogenic lines are genetically very similar, but differ in thepresence of a trait confering resistance to the fungal pathogenVerticuillium albo-atratum.

The first step of the modification of the DNAs comprised the restrictionof the DNAs with the two enzymes PstI and MseI. The restriction of theDNA, and also the subsequent ligation of the adapters to theDNA-fragments was carried out in the same buffer, which was namedRL-buffer (restriction-ligation buffer), and which contained: 10 mMTris.HAc/10 mM MgAc/50 mM KAc/5 mM DTT, pH 7.5.

Restriction of the DNAs with PstI and MseI

2.5 μg DNA

12.5 units PstI (Pharmacia, 10 units/μl)

12.5 units MseI (N.E. Biolabs, 4 units/μl)

5 μl 10×RL-buffer

H₂0 to 50 μl

Incubation was carried out at 37° C. for 1 hr.

The next step in the modification of the DNAs was the ligation oradapter molecules to the ends of the DNA fragments. First appropriatedouble-stranded adapter molecules had to be prepared.

Preparation of Adapters

MseI-adapter: 5-GACGATGAGTCCTGAG-3 (SEQ ID NO: 16) 3-TACTCAGGACTCAT-5(SEQ ID NO: 17)For preparation of a solution of 50 pMoles/μl of this adapter 8 μg (1430pMoles) of the 16-mer 5-GACGATGAGTCCTGAG-3 was mixed with 7 μg (1430pMoles) of the 14-mer 5-TACTCAGGACTCAT-3 in a total volume of 28.6 μl ofH₂0.

PstI-adapter: 5-bio-CTCGTAGACTCCGTACATGCA-3 (SEQ ID NO: 18)3-CATCTGACGCATGT-5 (SEQ ID NO: 19)For preparation of a solution of 5 pMoles/μl of this adapter 5.25 μg(715 pMoles) of the biotinylated 21-mer 5-bio-CTCGTAGACTGCCTACATGCA-3was mixed with 3.5 μg (715 pMoles) of the 14-mer 5-TGTACGCAGTCTAC-3 in atotal volume of 143 μl of H₂0.

Ligation of the Adapter Molecules

To the restricted DNA a mix of 10 μl was added containing:

1 μl PstI bio-adapter (=5 pMol)

1 μl MseI adapter (=50 pMol)

1.2 μl 10 mM ATP

1 10×RL-buffer

1 μnit T4 DNA ligase (Pharmacia, 5 units/μl)

H₂O to 10 μl

The resulting reaction mix of 60 μl was incubated for 3 hours at 37° C.The adapters were designed in such a way that the restriction sites werenot restored after ligation. In this way fragment-to-fragment ligationwas prevented, since fragment concatamers are restricted, because therestriction enzymes were still active during the ligation reaction.Adapter-to-adapter ligaton were not possible because the adapters werenot phosphorylated (see also example 1).

Selection of Biotinylated DNA-Fragments

Preparation of the template-DNAs for SRFA using two restriction enzymesgenerally involved an extra step not used when using SRFA with a singleenzyme. In this step the DNA-fragments to which a biotinylated adapterwas ligated wore separated from all other fragments.

Biotinylated fragments were separated from non-biotinylated fragments(MseI-MseI-fragments) in this step, by binding to paramagneticstreptavidine beads (Dynal). 10 μl beads were washed once in 100 μl STEX(100 mM NaCl/10 mM Tris.HCl/1 mM EDTA/0.1% Triton X-100 pH 8.0), andresuspended in 140 μl STEX. The beads were subsequently added to theligation mixture, to give a final volume of 200 μl. This was incubatedfor 30 minutes with gentle agitation at room temperature, to ensureproper binding of the biotinylated DNA-fragments to the beads. The beadswere collected by holding the tubes containing the beads close to amagnet. This prevented the beads from being pipetted when thesupernatant was transferred to another tube. The beads were washed once,and subsequently transferred to a fresh tube. Then the beads were washed3 times with 200 μl STEX. Finally the beads were resuspended in 200 μlTOl.E (10 mM Tris/0.1 mM EDTA, pH 8.0), and transferred to a fresh tube.The DNA was kept a 4° C.

The DNAs restricted with the restriction enzymes, provided withadapters, attached to the paramagnetic streptavidine heads and purifiedfrom the MseI-MseI fragments prepared as described above will bereferred to as template-DNAs in the following steps.

Amplification of PstI-MseI Fragments

The template-DNAs prepared as described above should contain allPstI-MseI fragments from the mentioned Tomato lines, and in addition asmall amount of PstI-PstI-fragments with no internal MseI-fragments. Inthis experiment a number of these PstI-MseI fragment s were visualisedby amplification, essentially as described in example 1. Gel analyses ofthe amplification products was performed on denaturing acrylamide gels(Maxam and Gilbert, Proc. Natl. Acad. Sci. U.S.A. 74, 560-564), becausethe kind of fragments obtained by the procedure described in thisexample were much smaller than the ones described in example 1. Inaddition these types of gels allowed the separation of up to 100 bandsper lane, which was about ten times more than the agarose gels describedin example 1. The fragments were visualised by labeling one of thePCR-primers at the 5′ end with (γ-³²P)ATP and polynucleotide kinase.

Labeling of the PCR-Primer

The primer selected for labeling was the 19-mer 5-GATGAGTCCTGAGTAAgaa-3(SEQ ID NO:20) which was named MseI-primer-1, and in which the selectivenucleotides are indicated with lower case letters. The labeling wasperformed in the following way:

3.0 μl 18-mer (from solution of 50 ng/μl=150 ng)5.0 μl (γ-³²P)-ATP (from solution of 10 μCi/μl=50 μCi)

3.0 μl 250 mM Tris.HCl/100 mM MgCl₂/50 mM DTT, pH 7.5

0.5 μl T4-kinase (Pharmacia 10 units/μl)

18.5 μl H20

This gave a total volume of 30 μl, which was incubated at 37° C. for 30minutes. For each PCR 1 μl of this 5′ labeled primer was added.

A total of 28 PCRs were performed, in which each of the 4 template-DNAswere amplified with 7 primer combinations. Each primer combination hadthe same MseI-primer (MseI-primer-1, described above), but varied in thechoice of the PstI-primer. A total of 7 different primers were chosen(As with the MseI-primer the selective nucleotides are indicated withlower case letters):

PstI-primer-1 (SEQ ID NO: 21): 5-CACTCCCTACATCCAGga-3 PstI-primer-2 (SEQID NO: 22): 5-GACTGCGTACATGCAGgt-3 PstI-primer-3 (SEQ ID NO: 23):5-GACTGCGTACATGCAGgg-3 PstI-primer-4 (SEQ ID NO: 24):5-GACTGCGTACATGCAGag-3 PstI-primer-5 (SEQ ID NO: 25):5-GACTGCGTACATGCAGat-3 PstI-primer-6 (SEQ ID NO: 26):5-GACTGCGTACATGCAGct-3 PstI-primer-7 (SEQ ID NO: 27):5-GACTGCGTACATGCAGta-3All PCR-primers were dissolved in H20 at a concentration of 50 ng/μl.

The Amplification Reaction

The PCR-mixture consisted of:2.0 μl of template-DNA1.0 μl of 5′ labeled MseI-primer (5 ng)0.5 μl unlabeled MseI-primer (25 ng)0.6 μl PstI primer (30 ng)

2.0 μl of 100 mM Tris.HCl/15 mM MgCl₂/500 mM KCl, pH 8.5

0.8 μl of 5 mM dNTPs0.1 μl of Taq polymerase (Cetus Perkin Elmer, 5 units/μl)

13.0 μl of H20

All components of the reaction were added and mixed well, an essentialcomponent of the PCR, generally the enzyme, was added last. Subsequentlythe reaction was started as soon as possible.

The amplifications were performed on a Perkin Elmer 9600 thermal cycler.The cycle profile was as follows:

 1 cycle: denaturation: 30 sec at 94° C. annealing: 30 sec at 65° C.extension: 60 sec at 72° C. 11 cycles: denaturation; 30 sec at 94° C.lower annealing temperature 0.7° C. each cycle, 64.3° C., 63.6° C.,62.9° C., 62.2° C., 61.5° C., 60.8° C., 60.1° C., 59.4° C., 58.7° C.,58.0° C., 57.3° C. incubate for 30 seconds at each temperature.extension: 60 sec at 72° C. 23 cycles: denaturation: 30 sec at 94° C.annealing: 30 sec at 56° C. extension: 60 sec at 72° C.

Gel Analysis of Amplified Fragments

The reaction products were analysed on 4.5% denaturing polyacrylamidegels. 50×38 cm gels were used, of which the gel cassettes to preparethese gels were purchased from Biorad. 100 ml of gel solution was usedcontaining 4.5% w/v acrylamide/0.225. % w/v bisacrylamide/7.5 M Urea/50mM Tris/50 mM Boric acid/1 mM EDTA, pH 8.3. 100 ml gel solution wasmixed with 500 μl 10% Ammonium persulfate and 100 μl TEMED immediatelybefore casting the gel. A Tris/Boric acid/EDTA-buffer was used aselectrophoresis buffer and contained: 100 mM Tris/100 mM Boric acid/2 mMEDTA, pH 8.3. The reaction mixtures were mixed with an equal volume (20μl) of 98% formamide/10 mM EDTA/0.01% w/v bromo phenol blue/0.01% w/vxylene cyanol. The resulting mixtures were heated for 3 minutes at 95°C., and then quickly cooled on ice. 2 μl of each sample was loaded onthe gel. Gels were run at constant power of 110 Watts to give a constantheat development during electrophoresis. Under these conditions thefield strength of the gels corresponded to 40 to 50 Volt/cm.

The results of the SRFA reactions are shown in FIG. 14. The lanes arenumbered from 1 to 28, and contain each time the tour Tomato lines withone of the 7 primer combinations. The order of the Tomato lines on thegel is: 1. GCR26, 2. GCR151, 3. Gem^(R), 4. Gem^(S).

Lanes 1 to 4 contain these DNAs amplified with MseI-primer-1 andPstI-primer-1, lanes 5 to 8 contain these DNAs amplified withMseI-primer-1 and PstI-primer-2, lanes 9 to 12 contain these DNAsamplified with MseI-primer-1 and PstI-primer-3, lanes 13 to 16 containthese DNAs amplified with MseI-primer-1 and PstI-primer-4, lanes 17 to20 contain these DNAs amplified with MseI-primer-1 and PstI-primer-5,lanes 21 to 24 contain these DNAs amplified with MseI-primer-1 andPstI-primer-6, and lanes 25 to 28 contain these DNAs amplified withMseI-primer-1 and PstI-primer-7. The gel contains no size markers butthe DNA fragments visualised correspond with ±200 nucleotides at thebottom of the Figure to ±500 nucleotides at the top.

Example 3 Selective Restriction Fragment Amplification of DNA of VariousLactuca Species with Two Restriction Enzymes

In example 2 the principle of selective restriction fragment (SRFA)amplification using two restriction enzymes is exemplified for TomatoDNA. In this example we will illustrate that similar results areobtained using DNAs of various Lactuca species using the same tworestriction enzymes PstI and MseI.

Isolation and Modification of the DNA

DNAs were isolated as described in example 1 using young leaf materialof various Lactuca species. As indicated below these plants include acommercial lettuce (L. sativa) variety, and several individuals of twowild Lactuca species, L. saligna and L. virosa. The plants werearbitrarily designated the following names:

1. L. saligna, nr. 21, plant 12. L. saligna, nr. 21, plant 23. L. saligna, nr. 22, plant 14. L. saligna, nr. 22, plant 25. L. virosa, nr. 01, plant 16. L. virosa, nr. 01, plant 27. L. virosa, nr. 02,8. L. virosa, nr. 03, plant 19. L. virosa, nr. 03, plant 210. L. sativa, a commercial butterhead varietyThe genetic material analysed thus represented 6 different plant types,including two different individuals of 4 of these plants.

Modification of the Lactuca DNAs to generate the templates for the SRFAwas performed identical to the procedure described in example 2.

Amplification of PstI-MseI Fragments

The DNAs prepared as described above were used as templates for SRFAreactions. Two primer combinations were used employing a singleMseI-primer and two different PstI-primers. These primers (selectivenucleotides depicted in lower case letters) were:

MseI-primer (SEQ ID NO: 28): 5-GATGAGTCCTGAGTAAaca-3 PstI-primer-1(SEQID NO: 29): 5-GACTGCGTACATGCAGaa-3 PstI-primer-2(SEQ ID NO: 30):5-GACTGCGTACATGCAGca-3

Amplification of PstI-MseI fragments using the primers depicted abovewas carried out exactly as described in example 2, and the generatedfragments were visualised on denaturing polyacrylamide gels as describedin example 2. The band patterns obtained are shown in FIG. 15. Lanes 1to 10 show DNAs 1 to 10 amplified with the MseI-primer in combinationwith PstI-primer-1, lanes 11 to 20 show lines 1 to 10 amplified with theMseI-primer in combination with the PstI-primer 2. Size markers (notvisible in this Figure) in nucleotides are indicated to the right of thegel. The differences in band patterns reflects the differences inrelatedness of the various plants.

Example 4 Selective Restriction Fragment Amplification of Corn InbredLines with a Variety of Restriction Enzyme Combinations

In example 2 and 3 the principle of selective restriction fragment(SRFA) amplification using two restriction enzymes is exemplified usingTomato DNA and Lettuce (Lactuca species) DNAs respectively. In thisexample it will be illustrated that similar results are obtained withCorn (Zea mais) lines. In addition it will be illustrated that a varietyof restriction enzyme combinations can be used to obtain DNAfingerprints of in this case Corn lines.

Isolation and Modification of the DNA

Two corn inbred lines were used, named 1 and 2. The source of theselines is irrelevant, because in our experience any selected line gavegood DNA fingerprints using SRFA. DNA of these lines was isolated fromyoung leaf material as described by Saghai-Mahoof et al. (1984), Proc.Natl. Acad. Sci. U.S.A. 81, 8014-8018). The following restriction enzymecombinations (EKs) were used to make the template-DNAs: PstI/TaqI,EcoRI/TaqI, AseI/TagI, Sse8387-I/TaqI. All enzymes were purchased fromPharmacia, except AseI which was purchased from New England Biolabs, andSse8387-I which was purchased from Amersham. Template DNAs were preparedessentially as described in examples 2 and 3, with the followingexceptions:

Restriction of the DNA was performed by first incubating with TaqI at65° C. for one hour, and subsequently incubating with the second enzyme,PstI, AseI, EcoRI or Sse8387-I, for an additional hour at 37° C.Ligation of adapters was as described in example 2 using the followingadapters:

(SEQ ID NOS: 31-32) TaqI-adapter: 5-GACGATGAGTCCTGAC-33-TACTCACCACTGGC-5 (SEQ ID NOS: 33-34) PstI & Sse8387-I-adapter: 5-bio-CTCGTAGACTGCGTACATGCA-3 3- CATCTGACGCATGT-5 (SEQ ID NOS: 35-36)AseI-adapter: 5-bio-CTCGTAGACTGCGTACC-3 3- CTCACGCATGGAT-5 (SEQ ID NOS:37-38) EcoRI-adapter: 5-bio-CTCGTAGACTGCGTACC-3 3- CTGACGCATGGTTAA-5

Amplification of Restriction Fragments

Amplification of restriction fragments was performed as described inexample 2. The primers selected for labeling of the amplificationproducts were the following TaqI-primers having 3 selective nucleotides(indicated by lower case letters):

TaqI-primers 1. 5-TGAGTCCTGACCGAacc-3 (SEQ ID NO: 39) (5′ labeled) 2.5-TGAGTCCTGACCGAaca-3 (SEQ ID NO: 40) 3. 5-TGAGTCCTGACCGAcaa-3 (SEQ IDNO: 41) 4. 5-TGAGTCCTGACCGAcaa-3 (SEQ ID NO: 42)These 4 primers were used for detection of amplification products withall four enzyme combinations. For each enzyme combination 4 primers forthe other enzyme were selected to give a total of 16 combinations foreach enzyme. These primers are indicated below (selective nucleotidesshown in lower case letters). For EcoRI and AseI primers with 3selective nucleotides were selected, for PstI primers with 2 selectivenucleotides were chosen, and for SseI primers with a single selectednucleotide were chosen. For enzymes cutting less frequently in the Corngenomic DNA, primers were selected containing extensions with fewerselective nucleotides.

EcoRI-primers: 1. 5-CTGCGTTACCAATTCcaa-3 (SEQ ID NO: 43) 2.5-CTGCGTTACCAATTCaca-3 (SEQ ID NO: 44) 3. 5-CTGCGTTACCAATTCaac-3 (SEQ IDNO: 45) 4. 5-CTGCGTTACCAATTCcag-3 (SEQ ID NO: 46) AseI-primers: 1.5-GACTGCGTACCTAATaac-3 (SEQ ID NO: 47) 2. 5-GACTGCGTACCTAATaag-3 (SEQ IDNO: 48) 3. 5-CACTGCGTACCTAATacc-3 (SEQ ID NO: 49) 4.5-GACTGCGTACCTAATgaa-3 (SEQ ID NO: 50) PstI-primers: 1.5-GACTGCGTACATGCAGac-3 (SEQ ID NO: 51) 2. 5-CACTGCGTACATGCAGaa-3 (SEQ IDNO: 52) 3. 5-GACTGCGTACATGCAGca-3 (SEQ ID NO: 53) 4.5-GACTGCGTACATCCACcc-3 (SEQ ID NO: 54) Sse8387-I-primers: 1.5-CACTGCGTACATGCAGGa-3 (SEQ ID NO: 55) 2. 5-GACTGCGTACATGCAGGg-3 (SEQ IDNO: 56) 3. 5-GACTGCGTACATCCACCc-3 (SEQ ID NO: 57) 4.5-GACTGCGTACATGCAGGt-3 (SEQ ID NO: 58)

A total of 128 PCRs were performed (2 DNAs×4 enzyme combinations×16primer combinations), following the protocol described in example 2. Thereaction products of these PCRs were analysed on 3 gels (containing 48lanes/gel) as described in example 2. All primer combination gave DNAfingerprints of 50 to 100 bands per lane, except for the combinationSseI/TaqI, which gave only 10 to 15 bands per lane. An example of one ofthe gels is shown in FIG. 16. This Figure shows part of the gel with theanalysis of DNA fingerprints obtained with the enzyme combinationsPstI/TaqI and EcoRI/TaqI. Lanes 1 to 8 show DNA-fingerprints of the twocorn DNAs obtained by SRFA with TaqI-primer-3 and PstI-primers-1, -2, -3and -4 respectively, lanes 9 to 1.6 show DNA-fingerprints of the twoCorn DNAs obtained by SRFA with TaqI-primer-4 and PstI-primers-1, -2, -2and -4 respectively, lane 17 shows the size marker lambda-DNA restrictedwith PstI, of which the sizes of some of the fragments in nucleotidesare indicated at the right, and lanes 18 to 25 show DNA fingerprints ofthe two Corn DNAs obtained by SRFA with TaqI-primer-4 and U-primers-1,-2, -3 and -4 respectively.

Example 5 Selective Restriction Fragment of Bacterial DNAs

In example 2, 3 and 4 the principle of selective restriction fragment(SRFA) amplification using two restriction enzymes is exemplified forTomato, Lettuce (Loctuca species) and Corn DNAs respectively. In thisexample it will be illustrated that this technique can also be used tocharacterize bacterial DNAs. A number or Xanthomonas campestris strainswere obtained from the Laboratory of Microbiology in Cent, Belgium, toillustrate the usability of the technique in bacteria.

Isolation and Modification of the DNA

All DNAs were prepared from Xanthomonas campestris strains isolated froma variety of origins, mostly from infected plants. These strains,numbered 1 to 26 are listed below, and may be obtained from theLaboratory of Microbiology in Ghent, Belgium.

DNA Subspecies pathovar isolate 1. albilneans 494 2. Fragariae 708 3.oryzae oryzae 5047 4. maltophilia 5743 5. campectris campestris 958 6.campestris alfalfae 568 7. campestris coracanae 497 8. campestris Citri686 9. campestris Citri 8655 10. campestris Citri 9658 11. campestrisCitri 9181 12. campestris Citri 8657 13. campestris Citri 8654 14.campestris Citri 8650 15. campestris Citri 682 16. campestris Citri 68117. campestris Citri 9325 18. campestris Citri 9321 19. campestris Citri9176 20. campestris Citri 9671 21. campestris Citri 9665 22. campestrisCitri 9182 23. campestris Citri 560 24. campestris Citri 9167 25.campestris Citri 9175 26. campestris Citri 9160DNA of these bacterial strains was isolated as described by Marmur (J.Mol. Biol. 3, 208-218). The DNAs were restricted essentially asdescribed in example 4, with the exception that TaqI and ApaI werechosen as restriction enzymes. Ligation of adapters was as described inexample 4 using the following adapters (SEQ ID NOS:59-62):

TaqI-adapter: 5-GACGATGAGTCCTGAC-3 3- TACTCAGGACTGGC-5 ApaI-adapter:5-bio-TCGTAGACTGCGTACAGGCC-3 3- CATCTGACGCATGT-5

Amplification of Restriction Fragments

Amplification of restriction fragments was performed as described inexample 2. The primers selected for SRFA were the TaqI-primer (SEQ IDNO:63) 5-CGATGAGTCCTGACCGAg-3 (having one selective nucleotide indicatedin lower case letter), and the ApaI-primer (SEQ ID NO:64)5-GACTGCGTACAGGCCCg-3 (having one selective nucleotide indicated inlower case letter). The ApaI-primer was labeled at the 5′ end fordetection of the amplified fragments as described in example 2.

Each of the 26 DNAs was amplified using the primer set described above.Amplification conditions were as described in example 2, except that thelast 9 cycles of the PCR were omitted, because of the lower complexityof the DNAs compared to the plant DNA in examples 2, 3 and 4.

The DNA fingerprints obtained with the bacterial DNAs as described inthis example are shown in FIG. 17. Lanes 1 to 26 represent bacterialDNAs 1 to 26. The sizes of marker DNAs (not visible on the gel) innucleotides are indicated to the right of the gel. This figures showsclearly that the relatedness of the bacterial strains is reflected bythe similarity of the band patterns.

Example 6 Selective Restriction Fragment Amplifications of DNA ofVarious Animals with Two Restriction Enzymes

In the previous examples selective restriction fragment amplification(SRFA) was exemplified for plant DNA of various sources. Here weillustrate the efficacy of the procedure using random samples of DNAobtained from different domestic animals. The animal species tested are:Gallus domesticus (chicken); Susscrofa domestics L. (pig); Bos taurus(cow); Equus caballus (horse). Restriction enzymes used are Sse8387I andMseI.

Isolation and Modification of the DNA

DNAs were isolated from blood samples following procedures described byManiatis et al. (1982). DNA samples 1 to 3 (chicken), 4 to 7 (pig), 8 to11 (cow) and 12 to 15 (horse) were digested by restriction enzymesSse8387I and MseI. The DNA fragments were ligated to adapters asdescribed in example 2. Since the restriction enzymes Sse83871 and PstIgenerate compatible 3′ overhangs we could use the PatI- and MseI-adapterdescribed in example 2.

Amplification of Restriction Fragments

Template DNAs named above and prepared as described in example 2 servedas templates in SRFA reactions. The primer combinations used consistedof a single MseI-primer and different SseI-primers:

MseI-primer (SEQ ID NO: 65): 5-GATGAGTCCTGACTAAtac-3 Sse83871-primer-2(SEQ ID NO: 66): 5-GACTGCGTACATGCAGGaa-3 Sse83871-primer-2 (SEQ ID NO:67): 5-GACTGCGTACATGCAGGag-3Amplification of Sse8387I-MseI fragments using primer pairs describedabove was carried out using the protocol described in example 2.Reaction products were run on denaturing polyacrylamide gels alsodescribed in example 2. An autoradiograph showing fingerprints of theabove samples is shown in FIG. 18. Lanes 1 through 15 show fingerprintsof DNA 1 to 15 amplified with the MseI-primer paired withSse8387I-primer-1, lanes 16 through 30 show similar patterns obtainedwith the MseI-primer combined with Sse83871-primer-2. Differences infingerprints between animals of one species reflect heterogeneity inanimal populations; overall patterns are characteristic for a specificspecies.

In a particular embodiment the invention relates to a process for thecontrolled amplification of at least one part of a starting DNA whichcontains a plurality of restriction sites for a determined specificrestriction endonucleacc, and of which at least part of its nucleic acidsequence is unknown, which process comprises:

-   -   (a) digesting said starting DNA with said specific restriction        endonuclease to fragment it into the corresponding series of        restriction fragments which respectively comprise 5′ ends and 3′        ends;    -   (b) unless the 5′ and 3′ adaptors defined hereafter were already        in separate forms, also digesting with said specific        endonuclease, a determined double-stranded oligonucleotide        linker including itself a single site within its own nucleotidic        sequence for said specific endonuclease to thereby cleave said        linker in such 5′ and 3′ adaptors respectively;    -   (c) ligating the restriction fragments obtained from the        starting DNA at their 5′ and 3′ ends with said 3′ and 5′        adaptors respectively to thereby produce tagged restriction        fragments of the starting DNA, which fragments then comprise at        their respective 5′ and 3′ ends tags whose nucleotide sequences        then comprise those of the 3′ and 5′ adaptors including the        nucleotides involved in the specific restriction site;    -   (d) unless, where appropriate to provide suitable templates for        primers, said 5′ and 3′ adaptors were prior to the preceding        ligation prolonged by adding thereto oligonucleotide segments of        determined constant sequences at their respective 5′ and 3′        ends, prolonging, where appropriate for the same purpose, the        corresponding ends of said tagged restriction fragments with        said oligonucleotide segments, whereby tagged restriction        fragments elongated at both ends with said constant sequences        are obtained;    -   (e) contacting said tagged or, when appropriate, elongated        restriction fragments under hybridizing conditions with two        oligonucleotide primers;    -   (f) wherein said primers include sequences having the same        nucleotide sequence as the terminal parts of the strands of the        5′ and 3′ ends of said tagged or, when appropriate, elongated        restriction fragments, which are themselves complementary to the        strands acting as templates for said primers, said primers        respectively including the nucleotides complementary to those        involved in the formation of the site for said determined        specific restriction endonuclease in the template strand;    -   (g) amplifying said elongated restriction fragments hybridized        with said primers by PCR or similar techniques in the presence        of the required nucleotides and polymerase to cause further        elongation of the hybridized primers along those restriction        fragments of the starting DNA to which said primers initially        hybridized on their entire length, and    -   (h) identifying or recovering said last mentioned restriction        fragments.

In a particular embodiment of this process, the terminal nucleotide ofat least one of said primers in the direction of the elongation soughtcorresponds to the last of the nucleotides involved in the restrictionsite for said specific endonuclease, and which process comprisesidentifying or recovering the restriction fragments of said starting DNAwhich have been amplified.

In another particular embodiment of this process, at least one of saidprimers includes a selected sequence comprising a determined number (oneor several nucleotides) extending beyond the last of the nucleotidesinvolved in the restriction site for said specific endonuclease in thedirection of its own elongation within the corresponding restrictionfragments during the amplification step.

In a specific embodiment of the above-described process, double-strandedDNA-linker contains several sites for different specific endonucleaseswhich are all distinct from one another, which processes compriserepeating, on a same starting DNA the steps of the process defined abovewith one of these restriction endonucleases yet with another of saiddistinct specific endonucleases and upon using primers whose nucleotidesequences are selected as defined in the above description, yet withrespect to said other specific endonuclease.

The process described above or of the oligonucleotide of the invention,is appropriate, for the identification of polymorphisms in determinedDNAs originating from the same live species, e.g. genomic DNAs of amicrobial, plant or animal, including humans, or of fragments thereof,either among or relative to a corresponding determined DNA standard,which use comprises subjecting the DNAs under study to the process or tothe contact of the oligonucleotide in conditions allowing anamplification or elongation reaction, comparing the restriction patternsobtained starting from each of said DNAs and, optionally, of saidstandard DNA and relating the existence and, where appropriate, thelocalization of that DNA polymorphism to the differences observedbetween the sizes of the restriction fragments of the different DNAs.

The invention also relates to a fragmented DNA whose different fragmentshave sequences which all correspond to initial digests of theunfragmented starting DNA from which they are produced with a samedetermined specific endonuclease, characterized in that all of saidfragments were tagged at their 5 and 3′ ends respectively by determined3′ and 5′ adaptors corresponding to the cleaved part of a same startingDNA linker which initially included a single restriction site for saidspecific endonuclease, and optionally prolonged with determined constantsequences. The fragmented DNA can be in the form of a pattern ofmigration bands on a suitable support, e.g. gel support, in which itsfragments had initially been caused to migrate under the influence of anelectric field.

The fragmented DNA can also comprise end portions includingoligonucleotide characterized by the following composition, startingfrom the 5′ end

-   -   (i) a nucleotide ocquence (constant sequence) of at least 10        bases, but not longer than 30 bases, complementary to a        determined DNA sequence used as adaptor, immediately followed        by:    -   (ii) a nucleotide sequence complementary to the target site of a        specific restriction endonuclease used in step (a), in so far as        that nucleotide or part of it, is not comprised in (ii),        immediately followed by:    -   (iii) a nucleotide sequence of at least one nucleotide, but        shorter than 10 nucleotides selected, e.g. which is 1 to 5        nucleotides long.

The invention further relates to a kit for the fragmentation ofdetermined DNAs by at least one specific restriction endonuclease intofragments and analysis of these fragments which comprises:

-   -   the specific restriction endonuclease;    -   a double-stranded DNA oligonucleotide linker including itself a        single site within its own nucleotidic sequence for said        specific endonuclease to thereby cleave said linker in        corresponding 5′ and 3′ adaptors respectively, wherein said        double-stranded DNA linker had a sufficient size to provide 5′        and 3′ parts which may subsequently provide templates for the        PCR primers of this kit;    -   PCR primers which respectively comprise, on the one hand the        same sequences as the strands of the 5′ and 3′ adaptors        complementary to the strands subsequently acting as templates        for said primers wherein said primers further include the        nucleotides complementary to those which are involved in the        formation of the site for said determined specific restriction        endonuclease in the template strands;    -   if appropriate, oligonucleotide segments of determined        (constant) sequences for generating sites of sufficient length        for hybridization with said primers, for the elongation of the        5′ ends of said 5′ adaptors or the 3′ ends of said 3′ adapters        or both, prior to digestion of said linker by said specific        restriction endonuclease to produce said 5′ and 3′ adaptors        respectively, or alternatively for the elongation of the tagged        fragments obtained subsequent to the ligation of said 5′ and 3′        adaptors to the extremities of the fragments of the starting        DNA;    -   optionally a fragmented DNA standard corresponding to the        determined DNA subject to a fragmentation study, whereby the        fragments of said DNA standard were obtained by digesting it        with said specific endonuclease.

A particular embodiment of this kit is such that said oligonucleotidesegments for the elongation of both said 5′ and 3′ adaptors or 5′ and 3′ends of the tagged DNA fragments, have identical nucleotide sequences.

In another embodiment, the linker of the kit contains several respectiveunique sites for specific endonucleases all different from one another,said kit further including primers corresponding to each of the 3′ and5′ adaptors formed by cleavage of said linker with said differentspecific endonucleases respectively, wherein said primers arerespectively as defined in claim 8, in respect of the 3′ and 5′ adaptorswhich are produced in said linker by cleavage thereof by each of saidspecific endonucleases.

Also in a particular embodiment the kit comprise fragmented DNAstandards as defined above in respect of the corresponding specificrestriction endonucleases, wherein each of said fragmented DNA standardsis in respect of each of the determined specific restriction enzymes.

1. A primer, comprising (A) a nucleotide sequence that is complementaryto at least part of an adaptor sequence; (B) a nucleotide sequence thatis complementary to at least part of a restriction recognition site; and(C) a selective nucleotide sequence consists of one to 10 nucleotideresidues, may be random or specific, and wherein upon hybridization tosaid target DNA, said selective sequence selects a subset of DNAfragments from the target DNA for amplification or elongation.
 2. Amethod for identifying an amplified restriction fragment from anindividual, comprising: (A) digesting DNA obtained from an individualwith at least one restriction endonuclease to produce restrictionfragments; (B) ligating a double-stranded adaptor to an end of at leastone restriction fragment to produce a tagged restriction fragment; and(C) amplifying the tagged restriction fragment(s) with two primers atleast one of which comprises a sequence complementary to at least partof the adaptor; wherein the amplification reaction reduces thecomplexity of the individual's digested DNA and selectively generates asubset of amplified restriction fragments.
 3. The method of claim 2,wherein two double-stranded adaptors are ligated to either end of arestriction fragment.
 4. The method of claim 2, wherein the individual'sDNA is digested with two or more restriction endonucleases.
 5. A methodfor identifying an amplified restriction fragment from an individual,comprising: (A) digesting DNA from an individual with at least onerestriction endonuclease to produce restriction fragments; (B) ligatinga double-stranded adaptor to each end of at least one restrictionfragment to produce a tagged restriction fragment; (C) amplifying thetagged restriction fragment(s) with two primers each of which comprisesa sequence complementary to at least part of the adaptors wherein theamplification reaction reduces the complexity of the individual'sdigested DNA and selectively generates a subset of amplified restrictionfragments.